The mitochondrion is a dynamic membrane-bound organelle that undergoes fusion and division. The balance between these opposing events, which occur in a coordinated manner, is a key determinant of organelle size, number, and shape. Mitochondrial fusion and division are mediated by conserved dynamin-related GTPases including Mfn (mammals)/Fzo1p (yeast) for fusion and Drp1 (mammals)/Dnm1p (yeast) for division. Abnormalities in mitochondrial fusion and division are associated with many neurodegenerative diseases such as Charcot-Marie-Tooth neuropathy, dominant optic atrophy, Alzheimer's disease, Huntington's disease, and Parkinson's disease. Many of these diseases affect postmitotic neurons, which contain mitochondria along their long axons and branched dendrites. Understanding the pathogenesis of these diseases requires a deeper knowledge of the molecular mechanisms that mediate and coordinate mitochondrial fusion and division as well as the physiological functions of these events. The proposed research will uncover how mitochondria fuse (Aim 1), how mitochondrial fusion and mitochondrial division are coordinated (Aim 2), and how mitochondrial division controls mitochondrial distribution in postmitotic neurons (Aim 3). To study the molecular mechanisms underlying mitochondrial fusion in Aim 1, we have purified and biochemically characterized two yeast proteins that are required for mitochondrial fusion- Fzo1p GTPase and the Fzo1p- binding protein Ugo1p. Using these proteins, we have developed assays for GTP binding, GTP hydrolysis, and GTP-dependent membrane fusion. These novel assays will allow us to dissect the functions of Fzo1p GTPase and Ugo1p in mitochondrial fusion. In Aim 2, we will determine how mitochondrial fusion and division are coordinated. We have shown that the loss of Drp1 reduces Mfn1 and Mfn2 levels in Drp1-null mouse embryonic fibroblasts. We will determine how changes in Drp1 levels are translated into regulation of Mfn levels and mitochondrial fusion. In Aim 3, we will determine the physiological roles of mitochondrial division in neurons by deleting Drp1 from postmitotic neurons using the Cre-loxP system in mice. Preliminary data show that Drp1 loss induces alterations in mitochondrial distribution and neurodegeneration. We will determine how mitochondrial division controls organelle morphology and distribution in postmitotic neurons. Successful completion of the proposed studies will provide mechanistic insights into mitochondrial fusion, the coordination mechanism that balances mitochondrial fusion and division, and the physiological role of mitochondrial division in neurons. PUBLIC HEALTH RELEVANCE: Abnormalities in mitochondrial fusion and division are associated with many neurological disorders. To gain a better understanding of the pathogenesis of these diseases, we will investigate the molecular mechanisms and physiological functions of mitochondrial fusion and division.